Non-MHC-restricted T lymphocytes and immune regulation

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CLINICAL I M M U N O L O G Y

Newsletter 7

The R o l e o f l n t e r p h a s e C y t o g e n e t i c s as a P o t e n t i a l T o o l i n t h e Cytodiagnosi$ of Urinary Bladder Carcinoma Ricardo S. Cajulis and Denise Frias-Hidvegi Section of Cytopathology, Department of Pathology, Northwestern Uruversity, Chtcago, Illinois t is now well established that most human neoplasms, both benign and malignant, have karyotypic changes detectable with existing cytogenetic techniques, and these changes are central to the biologic behavtor of these neoplasms. However, because of technical reasons, most neoplasms that have been stud:ed are hematopo:et~c :n origin with only a relatively few solid tumors having been investigated. With the advent of mterphase cytogenetlcs, that is, cytogenetic analysis done on nonmetaphase cells (interphase cells and terminally differentiated cells), chromosomal analysis of solid neoplasms is now performed with fewer difficult:es. This will eventually result in the identification of nonrandom chromosomal changes in this group of neoplasms, thereby facilitalang our understanding of its biology. The applicatmn of specml techniques to cytopathology has facilitated its transformation from a screening procedure to a diagnostic tool used in the management of patients with both neoplastic and non-neoplastic lesions. Various techniques such as immunocytochemislry, flow cytometry, and electron microscopy are no longer limited to histopathology but are now roufinely done using cytologic samples. The reasons that these spec:al techniques make use of cytologic samples are the ease of procurement, availability of fresh material if needed, and the use of prevtously stained slides if fresh samples are not available. These inherent advantages of cytologic samples are explored by interphase cytogenet:cs. For interphase cytogenetics to work properly and be adequately interpreted, specimens must be prepared in a monolayered fashion with abundant number of loosely cohesive fragments and :solated cells if possible. This important requirement is met when one uses fine needle asptrates (FNA), touch prepara-

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tions, and fluids from body cav:ties as well as smears prepared ex vivo.

T h e application of special techniques to cytopathology has facilitated its transformation from a screening procedure to a diagnostic tool used in the management of patients with both neoplastic and non-neoplastic lesions.Various techniques such as immunocytochemistry, flow cytometry, and electron microscopy are no longer limited to histopathology but are now routinely done using cytologic samples.

At Northwestern Memorial Hospital in Chicago, Illinois, we investigated the role of interphase cytogenetics in the cytodiagnosis of transitional cell carcinoma (TCC) in bladder washes. Since various reports in the literature have shown w:de ranges of sensitivity and specificity m detecting TCC by cytomorphology, and since DNAplo:dy analysis by flow cytometry (FCM) has been shown to increase its sensitivity, we performed a prospective study comparing conventional cytology, FCM, and interphase cytogenetics by fluorescence in situ hybridization (FISH) in 40 bladder washes with subsequent histologic confirmation. 3

Materials and Methods Forty bladder washes were collected in a clear plastic cup or bottle and tightly capped. Each sample was d:vided for flow

© 1994 Elsevser Science Inc.

cytomelric and cytologic evaluation. In the cytology laboratory, additional slides were prepared for FISH. Transurethral bladder biopsy for histologic evaluation was done in each case. Cytology and FISH Sample Preparation The sample was centrifuged for 10 min at 2,500 rpm in a cenlrifuge (Beckman TJ-6). Six slides were prepared for each case; 4 were stained using Papanicolaou's technique and 2 were set aside for FISH. The slides with the cell dispersions were dried for several minutes on a slide warmer. The sample was then fixed for 20 min in a coplin jar with methanol:acetic acid (3:1) chilled to-20°C. The slides were then Iransferred to a fresh fixative for an a&htiona120 min. The slides were then airdried and stored at-20°C until processing. Pretreatment of slides was carried out essentially with slight modification as m Cremer et al.~ and Hopman et al.~ Slides were wanned to room temperature prior to pretreatment. Slides were incubated for 5 mm at 37°C in 0.01% collagenase Blend H (Sigma Chemicals, Inc., St. Louis, MO) in Krebs Ringer, pH 7.0. Slides were then fixed in 1% formaldehyde solution in PBS for 10 rain at room temperature. The slides were washed in Dulbecco's PBS to remove residual formaldehyde, and then dehydrated through a 70%, 85%, and 100% alcohol step gradient, then air dried prior to hybridiTation. In situ Hybridization Hybridization and removal of unbound probe were carried out essentially as in Pinkel et al.9 The samples were incubated for 2 rain in a denaturant bath (70% formamide, 2X standard saline citrate (SSC), pH 7.6) at 70°C and were then dehydrated. To the warmed slides was apphed 10 lal of a hybridization solution containing Spec-

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Newsletter

tram Orange chromosome enumerator probe CEP 8 and CEP 12 (two separate slides for each probe). The chromosome enumerator probes and hybndiTation solution were provided by Imagenetics, Inc., Framingham, MA. The coverslipped slides were placed in a moist, sealed chamber, and incubated overnight at 42°(2 (16-20

hr). The following day the slides were washed three times for 10 mm each in a solution of 50% formamide with 2X SSC, pH 7.0, at 450C. The slides were then washed for 10 min each in 1X SSC, pH 7.0, and 2X SSC, 0.1% NP-40 at 45°C. The slides were removed from the bath, and carefully drained. The samples were counterstained wah 1 lag/ml DAPI (4, 6djamidino-2-phenyhndole) in an antioxidant mounting fluid prior to viewing. Two-Color FCN Sample Preparation Fresh bladder wash specimens were concentrated by centrifugation, resuspended in phosphate-buffered saline (PBS), and nucleated cells counted (at least 0.5 x 106 cells were required for two-color analysis). The details of the procedure are described elsewhere.3 Cytologic Evaluation All Papanicolaou-stained slides were evaluated for cytomorphologic evidence of transitional cell carcinoma (TCC). Each case was categorized as benign, reactive (negative for malignancy), or malignant (poslfive for TCC). FISH Evaluation The slides were viewed at 1000x magnification on a Zeiss Axiophot Fluorescence Microscope (Carl Zeiss, Inc., Oberkochen, West Germany), using a dual excitation/emission filter specific for DAPI and Spectrum Orange. The first 200 evaluable nucle~ in each case were analyzed. Evaluatmn and counting of FISH signals were performed according to criteria proposed by Hopman et al.6 A sample is considered positive for malignancy if 5% or more (_>10 nuclei) of the 200 nuclei counted showed aneuploidy. Aneuploidy is defined, in the present study, as the presence of at least 10 nucle~ with either one or more than two signals. If the above criterion for aneuploidy was not met, the case 019%1859/94/$0 00 + 7 00

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was considered diploid and therefore benign. FCM Evaluation All specimens must have a GdG~ peak coefficient of variation (CV) of no more than 7% for the histogram to be considered interpretable. Criteria for a positive (malignan0 bladder wash are similar to those described by Bachlament et al.2 Any specimen demonslrating DNA aneuploidy (i.e., two or more distinct GdGs peaks, each con-

Using histology as the gold standard, the overall concordance rate between histology and cytology, FCM, and FISH was 75%, 74%, and 83%, respectively .... The overall sensitivity of cytology was 61.5% .... FCM detected the presence of malignant cells in 15 of 21 evaluable samples for a sensitivity of 71.4% .... Interphase cytogenetics by FISH detected the presence of aneuploidy in 19 of 26 malignant samples for a sensitivity of 73%.

taining at least 5% of the total counts) is considered "positive for malignancy." In cytokeratin-gated specimens, a hyperdiploid fraction of at least 16% was also considered "positive for malignancy." Specimens with hyperdiploid fractions of 11 to 15.9% were reported as "suspioous for malignancy,"and those with less than 11% "negative for malignancy."For statistical purposes, the cases reported as suspicious for malignancy were categorized as "positive for malignancy" m the present study. Results The subsequent histologic evaluation of the biopsy materials revealed 26 transitional cell carcinomas (three Grade I, six Grade II, and 17 Grade III) and 14 benign lesions (eight cystitis, two cystitis w~th foO 1994 Elsevier Sctence Inc

cal fibroms, two Brunn's Nests, one postoperative spindle cell nodule, and two norreal tissue). Using histology as the gold standard, the overall concordance rate between histology and cytology, FCM, and FISH was 75%, 74%, and 83%, respectively. Cytology correctly diagnosed all the benign lesions as either benign or reactive transitional cells for a specificity of 100%. However, there were seven histologically Mgh-grade (Grades 2 and 3) and three lowgrade TCC not detected by cytology. Reevaluation of the slides revealed no cytologic evidence of either high- or lowgrade TCC following strict criteria in the cytodiagnosis of TCC. The overall sensitivity of cytology was 61.5%. FCM detected the presence of malignant cells m 15 of 21 evaluable samples for a sensitivity of 71.4%. These included the samples showing aneuplold and hyperdiploid (>11%) cell populations. Interestingly, two low-grade TCC showed aneuploid cell population. The six TCC not detected by FCM were all high grade. Nine of 40 cases (22.5%) were not evaluable due to insufficient number of cells for analysis. There were two falsepositive cases for a specificity of 80%. These cases showed histologic findings consistent with cystitis and focal submucosal fibrosis, which by FCM showed aneuploid cell populations. Interphase cytogenetics by FISH detected the presence of aneuploidy in 19 of 26 malignant samples for a sensitivity of 73% when two chromosome-specific probes were used (probes for chromosomes 8 and 12). However, the sensitivity dropped to 69% and 62% ffonly the probe for chromosome 8 or chromosome 12, respectively, was separately considered. The number of nuclei with abnormal signals ranged from 13 to 148, with an average of 64. The number of signals ranged from 1 to 9 per nucleus. The false-negative cases were two Grade I, one Grade II, and three Grade III TCC. Reevaluation of the six cases (slides for FISH) failed to meet the criteria for malignancy. There were no false-posiuve cases. Although some cases showed nuclei with one signal, none met the criteria of malignancy. The specificity of FISH, therefore, was 100%.

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Discussion Although the role of cytology in the detection of TCC of the urinary bladder is well established, well recognized problems arise m differentiating low-grade TCC from benign reactive transitional cells. The problem is inherent to the cytomorphoiogy since the cytologic changes in reactive conditions can mirmc a low-grade TCC. In addition, cytologic changes due to chemotherapy and radiation can pose additional problems to the cytopathologist in the cytodiagnosis of TCC. Wah the advent of special techniques such as FCM, the diagnosis of TCC in bladder washes has been facilitated. However, Badalament et al. concluded that although FCM is more sensitive than conventional cytology, it is not as specific.2 One major disadvantage of FCM is that it requires numerous cells for proper interpretation. Another consideration with the use of FCM as an adjunct in the detection of malignant cells is its correlation with conventional cytogenetic studies with respect to the presence or absence of aneuplo~dy. There are a number of reported discrepanc~es between the results of conventional cytogeneUc studies and FCM in the study of bladder neoplasm.6,t t-t 3,15 Although most of these studies have shown a good correlation between FCM and conventional cytogenetics with respect to detection of aneuploid cell population, there have been a number of cases that were aneuploid by conventional cytogenetics but DNA-diploid by FCM. The clinical significance of flus discrepancy is yet to be determined since only a few studies have been reported, mainly because of the difficult and tedious procedures involved in conventional cytogenetics in sohd tumors. In the recent past, interphase cytogenetics, i.e., cytogenetic analysis of terminally differentiated cells or cells in the interphase stage of the cell cycle, has been applied to a variety of neoplasms. In this study, interphase cytogenetics by FISH using chromosome-specific probes 8 and 12 showed concordance rates of 83%, 83%, and 84% with histology, cytology, and FCM, respectwely. Its sensitivity, 73%, is higher than that of cytology and FCM, 61.5% and 71.4%, respecavely. Two of

CLINICAL I M M U N O L O G Y

the three Grade I TCC were diploid by FISH. This is not unexpected since most low-grade TCC are diploid or hypodiploid by both conventional and interphase cytogenetic studies. ~.14 There were five histologically high-grade TCCs that were diploid by FISH. This observed discrepancy could be due to sampling or to the possibility that both chromosomes 8 and 12 were not altered numerically and therefore the presence of aneuploidy involving

T h e present study demonstrates the utility of interphase cytogenetics by FISH techniques as an adjunct in the diagnosis of TCC in bladder washes. Since a number of benign and reactive conditions can mimic TCC and thereby make conventional cytology equivocal in some instances, FISH can complement cytology in the cytodiagnosis and follow-up of patients with TCC.

other chromosomes cannot he ruled ouL TIc concordance rate for FISH and histology increased to 83% when two probes

were considered compared to 80% and 75% when probes 8 and 12 were considered separately. Thus, the use of more chromosome-specific probes increases the sensitivity of FISH in detecting the presence of aneupioidy. Although the concordance of cytology and FISH using only chromosome-specific probe 12 is 90%, this is not as meaningful as this concordance rate with histology (75%), which is considered the gold standard. The concordance rate for FCM and FISH is 84%. Numerical chromosome abnormalities observed by FISH on samples that are DNA diploid by FCM is not unexpected. The minimal detectable difference between a diploid cell population and an

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Newsletter 9

aneuploid population by FCM is dependent on both the proportion of aneuploid cells in the sample and the coefficient of variation (CV) of the GoG~ peaks. It becomes increasingly more difficult to detect an aneuploid population as its DNA content approaches 2c. Using FISH, it is possible to detect changes at the level of single chromosomes, but this is not possible by FCM. Even in the best scenario, with 50% of the cells being aneuploid and a 4% CV, the minimal detectable difference between the two populations would be 6 to 8%, or approximately three chromosomes. In general, when the two populations are present in equal proportions, bimodality (i.e., two separate, identifiable Go/G~peaks) is recognized only when the DNA content increase (or decrease) is approximately twice the CV) ° The high concordance rate (84%) between FISH and F C M with respectto the detectionof aneuploid tumor cells,despite methodologic differences,suggests that F C M can give an acceptableestimateof the totalchromosomal contentof a tumor cell. Currently, FCM analysis is still one of the most practical ways of determining DNA pioidy and SPF of tumor cells. Nevertheless, since DNA ploidy may help m predicting patient outcome in a number of neoplasms including TCC, FISH may complement FCM in refining these parameters especially in tumors that by FCM are diploid with low SPF. In instances wherein the samples are insufficient for FCM analysis, FISH may be used to determine the presence of aneuploidy. In this study, 9 of 40 (22.5%) cases did not have sufficient number of cells for FCM analysis. However, they had adequate number of cells for FISH. The present study demonstrates the utility of interphase cytogenetics by FISH techniques as an adjunct in the diagnosis of TCC in bladder washes. Since a number of benign and reactive conditions can mimic TCC and thereby make conventional cytology equivocal in some instances, FISH can complement cytology in the cytodiagnosis and follow-up of patients with TCC. FISH can also complement FCM especially in instances when FCM analysis is not feasible due to insuffi019%1859/94/$0.00 + 7.00

10 CLINICAL IMMUNOLOGY Newsletter

c~ent number of cells, as shown in thts study. Most importantly, interphase cytogenetics is a powerful tool that can be used to study various chromosomal abnormalities, both numerical and slructural, in mterphase cells, rendenng the cytogenetic analysis of solid tumors, including TCC, less techous and less time consuming. This technique will therefore facilitate our understanding of the biologic characteristics and behavior of TCC on a chromosome level in conjunction with conventional cytogeneUcs, ciN

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References 1

Babu VR, Lutz MD, Miles B J, et al • Tumor behavtor m transmonal cell carcinoma of the bladder m relalaonto chromosomal markers and lustopathology Cancer Res 47 6800--6805, 1987

2

Badalament RA, Gay H, Wintmore WF Jr, et al Momtonng mtravestcal Bacdlus CalmetteGuerm treatment of superficial bladder carctnoma by senal flow cytometry Cancer 58 2751-2757, 1986

3

Cajuhs RS et al Intesphase cytogenettcs as an

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adjunct m cytodtagnosts of unna W bladder carctnoma A comparaUve study of cytology, flow cytometry and mteq3hase cytogenettcs m bladder washes. Anal Quaut Cytol Htstol 16 1-10, 1994 Cremer T, Testa D, Hopman Aim, Manwehdts L Raptd mteq~hase and metaphase assessment of specific chromosomal changes m neuroectodermal tumor cells by m sttu hybndtzaUon wtth chemically modified DNA probes Exp Cell Res 176 199-200, 1988a. Coon JS, Schwartz D, Summers JL, et al. Flow cytometnc analysts of deparaff'unzed nuclet m urmaW bladder carcinoma Companson wtth cytogenettc analysts. Cancer 57.1594-1601, 1986 Hopmau A, Ramaekers FCS, Raap AIL et al In sttu hybndtzauon as a tool to study numencal chromosome aberratlons m solid bladder tumors Htstochenustry 89.307-316, 1988 Hopman A, Van Hooren E, Va de Kaa CA, et al DetecUon of numerical chromosome aberrauons using m sttu hybndtzatton m paraff'm secttons of routinely processed bladder cancer Mod Pathol 4 503-518, 1991 Pauwels RP, Smeets WW, Geraedts JP, Debroyne FM Cytogenettc analysts m urothehal cell carcinoma J Urol 136 210-225, 1987 Pmkel D, Landegent J, Coloms C, et al Fluorescence m s,tu hybndtzatton with human chromo-

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some-specafic hbranes' DetecUon of Tnsomy 21 and translocatton of chromosome 4 Proc Natl Acad S a USA 85.9138-9142, 1988 Rabmovttch PS: PracUcal constderauons for DNA content and cell cycle analysts In" Bauer KD, Duque RE, Shankey TV (eds) Chmcal Flow Cytometry Pluladelplua Wdhams and Wdkms, 1993, pp.117-142 Shackney SE, Burholt DR, Poihce AA, et al Dtscrepanctes between flow cytometnc and cytogenettc stuches m the detecuon of aneuplotdy on human sohd tumors Cytometry 11 94-104, 1990 TnbukaR B, Gnmberg-Ohmau I, Wukstrom H Flow cytomotnc DNA and cytogenettc studtes m human tumors: A comparison and dtscusston of the differences m modal values obtained by the two methods Cytometry 7'194-199, 1986 Waldman FM, Carroll PR, Kerschmann R, et al Centromenc copy number of chromosome 7 ts strongly correlated wtth tumor grade and labeling index m human bladder cancer Cancer Res 51 3807-3813, 1991 Wsjkstrom H, Granbeqg-Ohman I, TnbukaR B Chromosomal and DNA patterns m transmonal and bladder carcinoma. A comparaUve cytogenettc and flow-cytofluorometnc DNA study Caucer53 1718-1723, 1984

Non.MHC.Restricted T Lymphocytes and Immune Regulation Michael Grant I and Kenneth Rosenthal 2 llmmune Network Research Ltd , Vancouver, Br~t~sh Columbia, Canada, and 2Molecular V~rology and Immunology Program, Department of Pathology, McMaster Umversity, Hamilton, Ontarso, Canada

weU-founded regard for the central role of major histocompatibihty complex (MHC) molecules in selecting the T lymphocyte repertotre and in presenting antigens to T lymphocytes umtes the field of wnmunology. The MHCrestriction of antivtral cytotoxic T-cell responses, MHC-directed positive and negative selection in the thymus, and Xray crystallographic resolution of the molecular basis for MHC/T cell interactions rate among the most influential discoveries of modern immunology. A long hst of impressive publications introduced with pronouncements that "T lymphocytes exclusively recognize processed antigens

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presented as short pepUdes cradled within the ¢x-hehcal groove of self-MHC molecules" demonslrate the pervasive mapact of tins work. Few immunologists question the general validity of this statement, but we may have reached a point where the rare exceptions to this rule are more interestmg and reformative than its myriad examples. Taken literally, the fictmous, but highly representalave quote rendered above is unquestionably inaccurate. T-cell activation by superantigens, anti-T-cell receptor (TCR) antibodies, and by allogeneic cells debunks the myth of an absolute requtrement for either antigen processing or selfMHC in TCR-mechated activation of T

© 1994 Elsevier Scaence Inc

lymphocytes. The real issue is whether these recogmzed exceptions to the MHCrestriction rule reflect occult T-cell activitins with physiological relevance or a few curious anomalies. Some surprisingly well documented examples of non-MHC-restricted T cells inhabit the shadowy crypt of immunological heresy, but skeptics generally equate these w~th well documented examples of demonic visitation. Part of the skepticism or indifference to these reports stems from the reasonable supposition that tuning T lymphocytes to self-MHC as a condmon for thymic emigratton both inwgorates T cells and enforces harmony between the